JP2010117240A - Fluorescence detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence detection device capable of quantifying a biological material without being influenced by changes of the distance between an objective lens and a measurement object even in the case of using diffusion light as a light source. <P>SOLUTION: The fluorescence detection device is provided with a substrate for detecting a biological material having a measurement channel for measuring the biological material, and includes: a light source part for emitting diffusion light; an emitted-light suppressing slit disposed on the emission side of the light source part; a collimator lens disposed on the emission side of the emitted-light suppressing slit; a projection lens for condensing light transmitted through the collimator lens; an objective lens for projecting light emitted from the projection lens to the measurement channel; and a beam-shape suppressing slit disposed at a position conjugating with the imaging position of the light projected from the objective lens on the measurement channel. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a fluorescence detection apparatus that detects a biological material by detecting a transport reaction obtained by moving DNA, protein, or other biological material in a buffer.

  In recent years, research on analyzing biological materials has been actively conducted, and for example, progress in elucidation of DNA, RNA, proteins, etc. is remarkable. On the other hand, there is a demand for development of an apparatus that can quickly and accurately perform assay analysis of DNA and the like.

  These devices are used for the purpose of examining the presence or absence of a specific biological material and the expression of a mutant biological material. A biological material is detected using a DNA chip or a DNA microarray (hereinafter referred to as a biological material detection substrate) in which a biological material to be measured is fixed on a substrate such as glass or plastic. For detection of a biological material, a method using fluorescence by modifying a fluorescent label on the biological material is used. Since this amount of fluorescence is very weak, a highly sensitive fluorescence detection device is required.

  Examples of highly sensitive fluorescence detection devices include cooled CCD detection and confocal optical systems. In the system using a cooled CCD, weak fluorescence can be detected by increasing the exposure time. However, since the background noise is high, a confocal optical system is often used.

  The confocal optical system is an optical system that condenses fluorescence emitted from a fluorescently labeled biological material at a conjugate position, and performs detection using a photodiode or a photomultiplier tube installed at a condensing point. In a system using a confocal optical system, a scanning detection system of a laser-excited confocal optical system is generally used.

  A conventional fluorescence detection apparatus will be described with reference to FIG. This type of fluorescence detection apparatus uses a parallel light source 50 as a light source, and the light from the light source is reflected by a dichroic mirror 52 after passing through a light projection lens 51. The condensing point 53 of the light projecting lens 51 is optically adjusted so as to coincide with the front focal position of the objective lens 54, and the light passing through the objective lens 54 is irradiated to the specimen 55 as parallel light. The fluorescence emitted from the specimen 55 irradiated with the excitation light passes through the objective lens 54 and the dichroic mirror 52 and is imaged by the imaging lens 56 and detected by the detector 57 (see, for example, Patent Document 1). .

The biological material detection substrate 58 is made of glass, resin, or the like. However, since the dimensions cannot be made uniformly, slight distortion or deflection occurs. Therefore, when scanning the specimen in the biological material detection substrate 58, the distance between the objective lens and the specimen changes. In order to suppress the change in the beam size of the excitation light due to the change in the distance, the conventional photodetection device uses the light irradiated to the specimen 55 as parallel light. In this way, since the specimen is irradiated with parallel light, even when the distance between the objective lens and the specimen changes, the beam size and power of the light irradiated onto the specimen can be made uniform, and the excitation light is unevenly distributed on the measurement target. Irradiation was possible, and biological substances could be measured quantitatively.
JP 2002-357549 A

  However, in the above-described conventional configuration, since the light applied to the specimen is parallel light, the light source used is a light source that emits parallel light such as laser light. However, when laser light is used as a light source, there is a problem that the wavelength of the fluorescent label is limited because the laser wavelength is determined. Therefore, a configuration in which an arbitrary excitation filter is combined with a light source that emits diffused light such as a xenon lamp is conceivable.

  However, if a light source using diffused light is used in the conventional configuration, the light that has passed through the light projecting lens 51 cannot be collected at a single point, so that the light irradiated from the objective lens 54 to the specimen does not become parallel light. For this reason, when the distance between the objective lens 54 and the biological material detection substrate changes, the beam size of the light applied to the specimen changes. Along with this, the power density changes and the intensity of the excitation light applied to the specimen changes. When the excitation intensity is changed, the fluorescence detection amount is changed, so that there is a problem that a biological substance cannot be accurately measured.

  The present invention solves the above-described problems, and provides a fluorescence detection device that can quantitatively measure a biological material without being affected by a change in the distance between the objective lens and the measurement object even when diffuse light is used as a light source. The purpose is to provide.

  In order to solve the above-described conventional problems, the fluorescence detection device of the present invention includes a light source unit that emits diffused light in a fluorescence detection device that uses a biological material detection substrate provided with a measurement flow path for measuring biological material. , An exit light suppression slit disposed on the exit side of the light source unit, a collimator lens disposed on the exit side of the exit light suppression slit, a light projection lens that collects light that has passed through the collimator lens, and the light projection An objective lens that projects the light emitted from the lens onto the measurement flow path, and a beam shape suppression slit that is disposed at a position conjugate with the position at which the projection light of the objective lens forms an image on the measurement flow path. It is characterized by.

  According to the fluorescence detection device of the present invention, even if a change occurs in the distance between the objective lens and the measurement object, the amount of fluorescence of the biological material to be measured does not change. A fluorescence detection apparatus capable of quantitative measurement can be provided.

  Embodiments of the fluorescence detection apparatus of the present invention will be described below in detail with reference to the drawings.

(Embodiment 1)
FIG. 1 is a diagram showing a fluorescence detection apparatus according to Embodiment 1 of the present invention. This fluorescence detection apparatus is an apparatus for measuring fluorescence from a biological substance that is electrophoresed in a buffer solution.

  In this embodiment, a xenon lamp is used as the light source, and the xenon lamp connected to the optical fiber is the light source fiber 1. The light emitted from the light source fiber 1 passes through the outgoing light suppression slit 2 and the collimating lens 3. The light from the light source that has passed through the collimator lens 3 passes through the excitation filter 4 and the beam shape suppression slit 5. The excitation filter 4 is a filter for applying excitation light to the fluorescent label Cy5. The excitation filter 4 transmits the excitation wavelength region (594 nm to 651 nm) of the fluorescent label Cy5 and cuts the fluorescence wavelength region (669 nm to 726 nm) emitted by the fluorescent label Cy5. It has the property to do. The outgoing light suppression slit 2 and the beam shape suppression slit 5 which are features of the present invention will be described later.

  The light transmitted through the beam shape suppression slit 5 passes through the projection optical unit 16. In the projection optical unit 16, the light is converged by the light projecting lens 6 and reflected by the dichroic mirror 7. The dichroic mirror 7 has a characteristic of reflecting the excitation wavelength region (628 ± 20 nm) of the fluorescent label Cy5 and transmitting the fluorescence wavelength region (692 ± 20 nm) of the fluorescent label Cy5. The light reflected by the dichroic mirror 7 passes through the objective lens 8. The image forming position 17 of the light passing through the light projecting lens 6 and the front focal length of the objective lens 8 are arranged to coincide with each other. Thereby, the light passing through the objective lens 8 forms an image on the biological material detection substrate 14. In the present embodiment, the size of the projection light irradiated onto the biological material detection substrate 14 was 0.09 mm × 0.60 mm.

  If there is a sample in which the fluorescent label Cy5 is modified in the detection flow path 15 on the biological material detection substrate 14, fluorescence is excited from the sample by the projection light. The fluorescence is focused into parallel light by the objective lens 8 and passes through the dichroic mirror 7 and the fluorescence filter 10. As the fluorescent filter 10, a filter having a characteristic of transmitting light in the fluorescent wavelength region (692 ± 20 nm) of the fluorescent label Cy5 and cutting light from the light source is used.

The light transmitted through the fluorescent filter 10 is condensed at the position of the light shielding slit 12 by the imaging lens 11, and the amount of the specimen can be measured by receiving the light that has passed through the light shielding slit 12 by the detector 13. In addition, by setting the size of the light-shielding slit 12 to 0.09 mm × 0.60 mm, it is possible to shield stray light and suppress background noise.

First, the beam shape suppression slit 5 which is the first feature of the present invention will be described. FIG. 2A shows a schematic diagram of the beam shape suppression slit 5. In this example, the beam shape suppression slit 5 was made of a stainless steel substrate having an outer shape of 15 mm × 15 mm and a thickness of 0.2 mm. A rectangular hole 46 of 0.20 mm × 1.48 mm was provided at the center of the beam shape suppression slit 5. The position where the beam shape suppression slit 5 is disposed is important. This will be described with reference to FIG.

  FIG. 3 is a diagram showing a ray tracing diagram until the light emitted from the light source fiber 1 is projected onto the biological material detection substrate 14. For ease of explanation, the dichroic mirror 7 shown in FIG. 1 is omitted. The light emitted from the light source fiber 1 is projected onto the biological material detection substrate 14 through the collimating lens 3, the excitation filter 4, the beam shape suppression slit 5, the light projecting lens 6, and the objective lens 8. The beam shape suppression slit 5 must be provided at a position that is conjugate with the position at which the projection light from the objective lens 6 forms an image on the biological material detection substrate 14 as shown by the arrows in FIG. By arranging the beam shape suppression slit 5 at this position, the distance between the beam distance A′B ′ irradiated to the biological material detection substrate 14 and the distance AB of the rectangular hole 46 of the beam shape suppression slit 5 is as follows. The relationship holds.

AB / A'B '= L1 / L2
Here, L1 is the focal length of the projection lens 6, and L2 is the focal length of the objective lens 8. That is, the size of the rectangular hole 46 is determined by the size of the projection light, the focal length of the light projecting lens 6, and the focal length of the objective lens 8. In the case of Embodiment 1 of the present invention, the focal length of the projection lens 6 is 31 mm, and the focal length of the objective lens 8 is 14 mm. By setting the size of the beam shape suppression slit 5 to 0.20 mm × 1.48 mm, the size of the projection light can be about 14/31 (0.45) times 0.09 mm × 0.60 mm.

  Next, the outgoing light suppression slit 2, which is the second feature of the present invention, will be described. FIG. 2B shows a schematic diagram of the outgoing light suppression slit 2. In this example, the outgoing light suppression slit 2 was made of a stainless steel substrate having an outer shape of 15 mm × 15 mm and a thickness of 0.2 mm. A rectangular hole 45 of 0.76 mm × 6.00 mm was provided at the center of the outgoing light suppression slit 2. Since the position where this outgoing light suppression slit 2 is arranged is important, it will be described with reference to FIG.

  FIG. 4 is a diagram showing a ray tracing diagram until the light emitted from the light source fiber 1 is projected onto the biological material detection substrate 14. Like FIG. 3, the dichroic mirror 7 is omitted to make the explanation easy to understand. . The light emitted from the light source fiber 1 passes through the outgoing light suppression slit 2, the collimating lens 3, the excitation filter 4, the beam shape suppression slit 5, the light projection lens 6, and the objective lens 8, and is projected onto the biological material detection substrate 14. Is done. In this description, the front focal position of the objective lens 8 is referred to as a virtual plane 18.

  The outgoing light suppression slit 2 must be provided at a position having a conjugate relationship with the virtual surface 18 of the objective lens 8 as indicated by an arrow in FIG. By arranging the outgoing light suppression slit 2 at this position, the angle of the beam irradiated on the biological material detection substrate 14 can be made substantially parallel even if the light source is diffused light.

  Here, characteristics required for the projection light irradiated on the biological material detection substrate 14 will be described with reference to FIG. FIG. 5 shows the virtual surface 18, the objective lens 8, the biological material detection substrate 14, and light rays that pass through them. FIG. 5A shows the case where the distance between the surface 14a of the biological material detection substrate and the objective lens 8 is just focus, and FIG. 5B shows the surface 14b of the biological material detection substrate and the objective lens 8. The distance between and is defocused. 60a and 60b shown in FIGS. 5 (a) and 5 (b) respectively indicate the projection light sizes on the surface of the biological material detection substrate. As shown in FIG. 5, when the distance between the objective lens 8 and the biological material detection substrate 14 increases (defocused state), the projection light size becomes larger than the size 60a at the time of just focus. Thereby, the energy of the projection light per unit area becomes small, and the amount of energy of the light irradiated to the biological material detection substrate 14 decreases. As a result, the energy for exciting the fluorescent material decreases, and the amount of fluorescence detected decreases. Therefore, it is desirable that the projection light is as close to parallel light as possible.

  Therefore, in the present invention, the suppression emission light suppression slit 2 is provided at a position that is conjugate with the virtual surface 18 of the objective lens 8 as indicated by the arrow in FIG. By arranging the outgoing light suppression slit 2 at this position, as shown in FIG. 6, the following relationship is established between the focal length f, the position y ′ of the light beam, and the angle θ2 of the light beam.

y ′ = f · tan θ2
That is, the angle of the projection light is determined by the position of the light beam on the virtual surface 18 and the focal length of the objective lens 8. In the first embodiment, the angle of almost parallel light is designed to be 1.55 °. This 1.55 ° is an angle when the amount of change in the energy density of the projection light is reduced by 20% when the biological material detection substrate 14 is changed by 0.2 mm from the just focus position. By setting the size of the outgoing light suppression slit 2 to 0.76 mm × 6.00 mm, the angle of the projection light can be set to 1.55 °.

  Next, fluorescence detection from a biological substance that undergoes electrophoresis in a buffer solution using the configuration of the present invention will be described.

  FIG. 7A is a plan view showing the biological material detection substrate 14, and FIG. 7B is a diagram showing one flow path unit 21 in the enlarged view. The material of the biological material detection substrate 14 is an acrylic resin and has a thickness of 2 mm. A channel having a depth of 50 μm and a groove serving as a reservoir are dug on the channel forming surface, and a sealed channel is formed by adhering a 50 μm thick acrylic film to the upper surface. In addition, a hole for fixing the biological material detection substrate 14 to the fluorescence detection device is provided at the center of gravity 22. In the present embodiment, eight migration units 21 are arranged on the circumference of the biological material detection substrate 14 at regular intervals.

  The migration unit 21 of the biological material detection substrate 14 will be described in detail with reference to FIG. The sample in this example is a single-stranded DNA having a length of about 60 bases including a SNPs site to be discriminated, and is labeled with a fluorescent substance (Cy5). The specimen injected into the sample injection section 23 moves to the sample holding section 25 through the flow path 24, and a certain amount of specimen is held in the sample holding section 25. A sample exceeding a certain amount moves to the buffer unit 27 through the flow path 26.

  A DNA conjugate is provided as a buffer. The DNA conjugate is obtained by covalently bonding a high molecular linear polymer to the 5 ′ end of a 6 to 12 base long single-stranded DNA. Furthermore, DNA is a sequence that is complementary to the normal type but not complementary to the mutant type, has a strong binding force to the normal type DNA, and a weak binding force to the mutant type DNA. There are characteristics. In addition, when electrophoresed, it bound to the 5 ′ end. There is also a characteristic that the migration speed is considerably slow due to the weight of the linear polymer. The buffer injected into the buffer injection unit 28 moves from the channel 29 to the positive electrode unit 30 and the negative electrode unit 31, and further fills the channel 32 and the detection channel 15.

  A voltage is applied between the positive electrode part 30 and the negative electrode part 31. The voltage is applied by providing an electrode sheet so that the positive electrode part 30 and the negative electrode part 31 are in contact with the buffer, and the needle-like electrode is in contact with the electrode sheet. By applying a voltage, an electric field is generated in the flow path 32 and the detection flow path 15, and the specimen held in the sample holding unit 25 is electrophoresed in the detection flow path 15.

  By adopting the configuration of the present embodiment, even when a diffusion light source is used, it is possible to detect the light emission amount of the specimen that is to be electrophoresed in the detection flow path 15, and the case where a conventional parallel light source is used. Similarly, quantitative measurement was possible.

(Embodiment 2)
FIG. 8 shows a fluorescence detection apparatus according to Embodiment 2 of the present invention. In FIG. 8, the difference from Embodiment 1 is that it can respond to a plurality of biological substances having different excitation light.

  In this embodiment, an example using a filter wheel 35 is shown. FIG. 9 is a diagram showing a schematic diagram of the filter wheel 35. The filter wheel 35 includes a first excitation filter 36, a second excitation filter 37, and a third excitation filter 38. A rotation shaft 41 is provided at the center of the filter wheel 35.

  In the second embodiment of the present invention, the first to third excitation filters 36 to 38 have excitation wavelength regions (407/14 nm, 494/20 nm, 576/20 nm) of the fluorescence labels DAPI, FITC, and TRED, respectively. A band pass filter for transmission was used.

  In FIG. 8, the light emitted from the light source fiber 1 passes through the outgoing light suppression slit 2 and the collimating lens 3 and passes through the first excitation filter 36 attached to the filter wheel 35 to select the wavelength. . The light that has passed through the first excitation filter 36 passes through the beam shape suppression slit 5 and the light projecting lens 6 and is then reflected by the multi-multidic dichroic mirror 39.

  A multi-band bandpass filter was used for the multi-multi dichroic mirror 39. The characteristics of the multi-multi dichroic mirror 39 are shown in FIG. The multi-multi dichroic mirror 39 reflects light (407/14 nm, 494/20 nm, 576/20 nm) in the excitation wavelength region of the fluorescence labels DAPI, FITC, TRED, and light in the fluorescence wavelength region of the fluorescence labels DAPI, FITC, TRED. (457/22 nm, 530/20 nm, 628/28 nm).

  The light reflected by the multi-dichroic mirror 39 passes through the objective lens 8 and is projected onto the specimen on the biological material detection substrate 14. The fluorescence emitted from the specimen excited by the projection light is focused by the objective lens 8. The light passes through the multi dichroic mirror 39 and the multi fluorescent filter 40. The multi-fluorescence filter 40 transmits light (457/22 nm, 530/20 nm, 628/28 nm) of fluorescence labels DAPI, FITC, TRED, and light in the excitation wavelength range of fluorescence labels DAPI, FITC, TRED ( 407/14 nm, 494/20 nm, and 576/20 nm) were used. The light that has passed through the multi-fluorescent filter 40 is condensed at the position of the light shielding slit 12 by the imaging lens 11, and the light that has passed through the light shielding slit 12 is received by the detector 13.

  In the second embodiment, three types of specimens were electrophoresed simultaneously. Each specimen was previously modified with fluorescent labels DAPI, FITC, and TRED with different excitation light. Next, using the first excitation filter 36, fluorescence measurement of a biological substance labeled with the fluorescent substance DAPI was performed. Subsequently, the filter wheel 35 was rotated to switch from the first excitation filter 36 to the second excitation filter 37. At this time, the second excitation filter 37 is arranged so that the light passing through the collimating lens 3 is transmitted. Using the second excitation filter 37, the biological material modified with the fluorescent material FITC was measured. Similarly, a biological material labeled with the fluorescent material 38 was measured using the third excitation filter 38.

  Thus, by rotating the filter wheel 35 and switching the first to third excitation filters 36 to 38, three types of biological substances can be analyzed respectively.

  As described above, in the second embodiment of the present invention, when there are a plurality of biological substances to be discriminated simultaneously, the fluorescent labels DAPI, FITC, and TRED are respectively labeled on the biological substances to be discriminated, and the respective biological substances are analyzed. It is possible. In particular, by providing the outgoing light suppression slit 2 and the beam shape suppression slit 5, the biological material can be quantitatively measured without being affected by the change in the distance between the objective lens and the measurement object.

  The fluorescence detection apparatus according to the present invention is useful in that it can quantitatively detect a biological material even when an extended light source is used, and can measure a plurality of biological materials.

Configuration diagram of a fluorescence detection apparatus according to Embodiment 1 of the present invention Schematic diagram of beam shape suppression slit 5 and outgoing light suppression slit 2 in Embodiment 1 of the present invention Ray tracing diagram until it is projected onto the biological material detection substrate 14 in the first embodiment of the present invention. Ray tracing diagram until it is projected onto the biological material detection substrate 14 in the first embodiment of the present invention. Objective lens 8, biological material detection substrate 14, and projected ray tracing diagram in Embodiment 1 of the present invention Ray tracing diagram of lens 19 with focal length f Configuration diagram and migrating unit schematic diagram of biological material detection substrate in Embodiment 1 of the present invention Configuration diagram of a fluorescence detection apparatus according to Embodiment 2 of the present invention The block diagram of the filter wheel 35 in Embodiment 2 of this invention The figure which shows the characteristic of the multi dichroic mirror 39 in Embodiment 2 of this invention. Configuration diagram of conventional fluorescence detector

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fiber for light source 2 Emission light suppression slit 3 Collimating lens 4 Excitation filter 5 Beam shape suppression slit 6 Light projection lens 7 Dichroic mirror 8 Objective lens 10 Fluorescence filter 11 Imaging lens 12 Light-shielding slit 13 Detector 14 Biological substance detection board DESCRIPTION OF SYMBOLS 15 Detection flow path 16 Projection optical part 17 Imaging position 18 Virtual surface 19 Lens 20 Drive part 21 Migration unit 22 Center of gravity 23 Sample injection part 24 Flow path 25 Sample holding part 26 Flow path 27 Buffer part 28 Buffer injection part 29 Flow Path 30 Positive electrode 31 Negative electrode 32 Flow path 35 Filter wheel 36 First excitation filter 37 Second excitation filter 38 Third excitation filter 39 Multi dichroic mirror 40 Multi fluorescent filter 41 Rotating shaft 45 Rectangular hole 46 Rectangular hole 50 Parallel Light source 51 Light lens 52 dichroic mirror 53 focusing point 54 objective lens 55 specimen 56 imaging lens 57 detector 58 biological substance detection board 60a, 60b projected light Size

Claims (7)

In a fluorescence detection apparatus using a biological material detection substrate provided with a measurement channel for measuring biological material,
A light source that emits diffused light;
An outgoing light suppression slit disposed on the outgoing side of the light source unit;
A collimating lens disposed on the exit side of the exit light suppression slit;
A light projection lens that collects the light that has passed through the collimator lens;
An objective lens that projects the light emitted from the light projecting lens onto the measurement channel;
A beam shape suppression slit disposed at a position conjugate with a position where the projection light of the objective lens forms an image on the measurement channel;
A fluorescence detection apparatus comprising: The fluorescence detection apparatus according to claim 1, wherein the light source unit includes a light source that emits diffused light and an optical fiber, and emits diffused light of the light source from an end surface of the optical fiber. The outgoing light suppression slit is
The fluorescence detection apparatus according to claim 1, wherein the fluorescence detection apparatus is disposed at a position conjugate with a front focal position of the objective lens. The beam shape suppression slit is
2. The rectangular hole for shaping the light that has passed through the collimator lens so that the shape of the irradiation light on the measurement channel is rectangular and the longitudinal direction of the rectangle is perpendicular to the measurement channel. The fluorescence detection apparatus as described. The fluorescence detection device according to claim 4, wherein the size of the projection light on the measurement flow path is equal to a value obtained by multiplying a size of the rectangular hole by a quotient of a focal length of the projection lens and a focal length of the objective lens. . The fluorescence detection apparatus according to claim 1, wherein an excitation filter is provided between the collimating lens and the beam shape suppression slit. The fluorescence detection apparatus according to claim 1, wherein a filter wheel in which a plurality of excitation filters having different wavelengths are arranged radially is provided between the collimating lens and the beam shape suppression slit.

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